Transgenic corn plants having seeds with modified cornstarch characteristics and method of making the transgenic corn plants

ABSTRACT

Cornstarch characteristics can be changed by expressing a non-corn plant starch branching enzyme in a corn plant. In a preferred embodiment, transgenic corn plants containing the barley starch branching enzyme IIa transgene was generated. Some transgenic corn plants produced seeds containing cornstarch with lowered gelatinization temperature and retrogradation rate while others produced seeds containing cornstarch with higher retrogradation rate when compared to non-transgenic corn plants.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 60/295,649, filed on Jun. 4, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Starch is a polymer of glucose linked by α(1–4) linkages to give linearchains, H which are joined by α(1–6) linkages resulting in branches inthe polymer. Normal starch consists of about 75% amylopectin, ahighly-branched molecule, and 25% of amylose, a primarily linearmolecule. These polymers are organized into an insoluble granule withincereal seeds. Starch granules are highly organized, containing of aseries of concentric spheres consisting of alternating crystalline andamorphous regions (Cameron and Donald, 1992).

Starch is synthesized by a series of enzymatic reactions (for review,see Martin and Smith, 1995; Myers et al., 2000). Glucose-1-phosphate isfirst activated to ADP-glucose by the enzyme ADP-glucosepyrophosphorylase (ADPGPP). This enzyme is heavily regulated and isthought to control the flux of carbon into starch biosynthesis, andtherefore the amount of starch made. The structure of starch isdetermined by the subsequent enzymes in the pathway. Starch synthases(SS) catalyze the polymerization of ADP-glucose to produce a linearglucan polymer. Branches are introduced into this polymer by starchbranching enzymes (SBE). Starch debranching enzymes (SDBE) contribute tostarch structure by removing excess branches, which may help toestablish the pattern of crystalline and amorphous regions within thegranule.

Starch structures differ in different species. For example, barley andwheat amylopectins have larger portions of short branch chains (6 to 14glucose units), have proportionally fewer branch chains of 11 to 22glucose units and >40 glucose units, and larger proportions of branchlinkages located within the crystalline region than maize amylopectin(Jane et al., 1999; Song and Jane, 2000). It is the starch structurethat determines the functionality of starch.

The higher starch yield of corn as a C-4 crop makes cornstarch the mosteconomic commodity in the world. Starch is easily isolated from cornseeds during milling process as compared to barley or wheat whose awnsor gluten makes starch separation more difficult. In addition, thehigher phospholipid content of barley and wheat starches restrictsstarch swelling and paste viscosity. However, barley or wheat starcheshave lower gelatinization temperatures than cornstarch, and thus requireless energy for processing and cooking (Jane et al., 1999). Compared tocornstarch, barley or wheat starch has a lower retrogradation rateduring storage, which translates into better paste stability andprolonged shelf life (Shi and Seib, 1992,1995; Yuan et al., 1993; Janeet al., 1999). Barley and wheat starches are also easier to digest byenzyme and animals than cornstarch, which can result in faster glucoseproduction from starch and increased energy availability to livestock.This is particularly beneficial to young and small animals such as babychicks that have shorter digestive tracts.

Chemical modifications of starch (e.g. chemical derivatives) arecommonly used in the wet-milling industry to reduce the gelatinizationtemperature and retrogradation rate. Chemical modification processes areenergy demanding, requiring large quantities of chemical reagents andsalts during the reaction and washing and drying after the reaction.Recovery of byproducts, unreacted reagents, and salts (e.g. sodiumsulfate) from wastewater is costly and has the potential to causeenvironmental pollution.

Genetic modification of starch structure provides an attractivealternative to improve functionality. It was estimated that geneticmodification of cornstarch structure and functionality could add value$1.25 billion per year, with an average added value of $5.80 per bushel(Johnson et al., 1999a). An increase of cornstarch digestibility by 10%for livestock feed would add another $1.44 billion per year, with anaverage added value of $0.21 per bushel (Johnson et al., 1999b). Inaddition, genetically modified corn may be used to make possible newstarch products (such as biodegradable plastics) and create new markets.

Genes or cDNAs of most starch biosynthetic enzymes have been cloned incorn, potato, barley, and wheat. They can be used to over- orunder-express these enzymes by using sense or antisense transgenes.Expression of an E. coli ADPGPP in potato tubers increased starchcontent by 35% (Stark et al., 1992). Transformation of the amylosedeficient amf mutant of potato with the granule-bound starch synthase(GBSS) gene led to amylose synthesis (Flipse et al., 1994), whereasvarious levels of reduction in GBSS protein and amylose were observed intransgenic rice endosperm with the antisense GBSS gene connected to therice GBSS promoter or maize Adhl promoter (Terada et al., 2000).Antisense inhibition of two soluble SS of potato individually orsimultaneously led to distorted starch granules and an enrichment inshort chains and a reduction in longer chains of amylopectin (Edwards etal., 1999; Lloyd et al., 1999). Expression of an E. coli branchingenzyme in tubers of amylose-free potato showed an increased branchingdegree and more short chains (16 glucose-residues or less) of theamylopectin (Kortstee et al., 1996). The antisense inhibition of themain SBE in potato tubers (SBE B) resulted in novel starchcharacteristics but not in an increased amylose level (Safford et al.,1998). However, transgenic potato plants expressing an antisense SBE A(the minor form of SBE) RNA increased the average chain length ofamylopectin, resulting an moderate increase in apparent amylose contentup to 38% (Jobling et al., 1999). Antisense inhibition of both SBE A andB simultaneously led to the production of potato starch withhigh-amylose and no amylopectin (Schwall et al., 2000).

There is a need in the art for a method of producing cornstarchcombining the advantages and avoiding the disadvantages of corn andbarley starches using genetic engineering.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that cornstarch characteristicscan be changed by expressing a non-corn plant SBE in a corn plant. Inone aspect, the present invention is a transgenic corn plant thatcontains a polynucleotide transgene. The transgene encodes a proteinhaving a non-corn plant SBE activity wherein the expression of theprotein in a corn plant can change a cornstarch characteristic ascompared to a plant of the same genetic background without thepolynucleotide transgene. A transgenic plant cell or tissue including aseed that contains the polynucleotide transgene is also within the scopeof the present invention.

In another aspect, the present invention is a method of generating atransgenic corn plant wherein seeds from the transgenic corn plantcontain cornstarch with a changed characteristic. The method involvescontacting a corn plant cell with a nucleic acid that contains apolynucleotide encoding a protein that has a non-corn SBE activity,identifying a plant cell carrying the polynucleotide, and regenerating atransgenic plant from the plant cell identified. A plant obtained by themethod of the present invention and cells and tissues including seeds ofthe plant are also within the scope of the present invention.

It is an advantage of the present invention that the cornstarchcharacteristics are changed using genetic engineering so that potentialenvironmental pollution problems associated with chemical modificationsof starches in changing starch characteristics are avoided.

It is another advantage of the present invention that the method ofmodifying cornstarch characteristics is easy and cost effective incomparison to prior art chemical modification methods.

Compared with the starches of corn mutants affecting starch compositionin endosperm, starches from transgenic plant can provide a wide rangeand combinations of starch structure and functionality to meet needs ofthe food and industrial applications.

Other objects, advantages and features of the present invention willbecome apparent from the following specifications and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a map of the pMO5 plasmid.

FIG. 2 shows barley sbeIIa gene constructs and primers used to amplifythe transgene. The constructs have a maize γ-zein promoter (γ-zein P),first intron of maize adhl gene (I), full-length barley sbeIIa cDNA, andterminator region from A. tumefaciens nopaline synthase gene (Nos), andis built on a pUC19 backbone. Primers puc, zein1, and nos were used toamplify a 3.4 or 4.4 kb transgene product. Vector pMO12 has a 6×His-tagjust before the stop codon of the sbeIIa gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to using genetic engineering to changecornstarch characteristics, which involves generating transgenic cornplants that express a polynucleotide encoding a protein that has anon-corn plant SBE activity. A non-corn plant SBE activity is definedherein as the activity of an SBE from a plant other than a corn plant.The expression of the polynucleotide in a corn plant may changecornstarch characteristics in one of two ways. One, the non-corn plantSBE activity can cause cornstarch to have characteristics closer tostarch from a plant in which the non-corn plant SBE is nativelyexpressed. Two, when the expression level of the polynucleotide in thetransgenic plant is above a certain threshold level leading to silencingof all branching enzyme activities, cornstarch produced from thetransgenic plant will have a higher gelatinization temperature andretrogradation rate; the cornstarch will also have a higher amylosecontent and thus share functional characteristics of high-amylosestarch.

The present invention is illustrated in the example below with barleySBE IIa. Barley starch has a lower starch gelatinization temperature andretrogradation rate in comparison to cornstarch. Expressing barley SBEIIa in a corn plant led to lower starch gelatinization temperature andretrogradation rate in some corn plants and higher retrogradation ratein other corn plants.

To make a transgenic corn plant that expresses a polynucleotide encodinga non-corn plant SBE activity, as is known to those of skill in the art,one needs to make a genetic construction capable of expressing thepolynucleotide in a corn plant. One also needs a method to insert thegenetic construction into the plant.

The tools and techniques for making genetic constructions that willexpress proteins in plants are now widely known. Any geneticconstruction intended to cause the synthesis in the cells of the plantof a polypeptide or protein must include a sequence of DNA known as aprotein coding sequence (can be a genomic DNA or a cDNA), whichspecifies the sequence of the polypeptide or protein to be produced inthe resultant plant. For a protein coding sequence to be expressed in aplant to produce a polypeptide or protein, it must be placed under thecontrol of a plant expressible promoter and be followed by a planttranscriptional terminator sequence, also known as a polyadenlyationsequence. The plant expressible promoter is a promoter which will workin plants, usually either of plant origin or from a plant pathogen likea virus (e.g. Cauliflower mosaic virus) or a bacteria (e.g.Agrobacterium promoters like the nopaline synthase promoter).

Plant promoters from pathogens tend to be constitutive promoters,meaning that they actually express the protein coding sequence in all ofthe tissues of the plant at all times. Examples of constitutivepromoters useful in plant genetic constructions include, withoutlimitation, the 35S RNA and 19S RNA promoters of the Cauliflower mosaicvirus (Brisson et al., Nature, 310, 511, 1984), and the opine synthasepromoters carried on the tumor-inducing plasmids of Agrobacteriumtumefaciens such as the nopaline synthase promoter (Ebert et al., PNAS,84, 5745, 1987) and the mannopine synthase promoter (Velten et al., EMBOJ. 3, 2723 1984).

Other plant promoters are known to be tissue specific or developmentallyspecific, while others are intended to be inducible (e.g. heat shock ormetal ion induced promoters). An example of tissue specific promoters isthe maize γ-zein promoter used in the example below for expressing genesin endosperm. Endosperm specific promoters are preferred promoters forthe present invention. Examples of inducible promoters suitable for usein the present invention include, but are not limited to, heat shockpromoters such as soybean hsp17.5E or hsp17.3 (Gurley et al., Mol. CellBiol. 6, 559, 1986), light-regulated promoters such as the promoter forthe small subunit or ribulose bisphosphate carboxylase (ssRUBISCO)(Coruzzi et al., EMBO J. 3, 1671, 1984; Broglie et al., Science 224,838, 1984), chemical-regulated promoters such as Maize In2-1 and 2-2which are regulated by benzenesulfonamides, e.g., herbicide safeners(Hershey et al., Plant Mol. Biol., 17, 679, 1991), and alcA and alcRpromoter/transcription factor system that is induced by the applicationof ethanol (Caddick et al., Nat. Biotech., 16, 177, 1998).

Any of the promoters described above may be used in the practice of thisinvention depending on the intended effect on the transgenic corn plantto be produced. For example, adjusting the expression level of apolynucleotide encoding a non-corn plant SBE activity by varyingpromoter strength may determine the likelihood of the transgenic plantto have the non-corn plant SBE activity or to have all branching enzymeactivities silenced.

Optionally, a selectable marker may be associated with a geneticconstruct used to generate a transgenic plant. As used herein, the term“marker” refers to a gene encoding a trait or a phenotype which permitsthe selection of, or the screening for, a plant or plant cell containingthe marker. Preferably, the marker gene is an antibiotic resistance genewhereby the appropriate antibiotic can be used to select for transformedcells from among cells that are not transformed. Examples of suitableselectable markers include adenosine deaminase, dihydrofolate reductase,hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guaninephospho-ribosyltransferase, and amino-glycoside 3′-O-phosphotransferaseII (which confers kanamycin, neomycin and G418 resistance). Othersuitable markers will be known to those of skill in the art.

Several methods have been demonstrated to insert genes into plants tomake them transgenic. The most widely used methods, broadly defined, areAgrobacterium-mediated transformation and accelerated particle mediatedtransformation (as illustrated in the example below). The varioustechniques of Agrobacterium-mediated plant transformation make use ofthe natural ability of the plant pathogens of the Agrobacterium genus totransfer DNA from a plasmid in the bacteria into the genome of a plantcell. Particle-mediated plant transformation techniques utilizeDNA-coated small carrier particles accelerated from a device, oftenreferred to as a gene gun, into the cells of a plant. The fullimplementation of either approach requires techniques to recover a fullymature, morphologically normal plant from the transformed cells. Thetechniques often therefore involve either selection or screeningprotocols to identify which plant cell was transformed and regenerationprotocols to recover a whole plant from a single transformed plant cell.As mentioned above, these techniques have been worked out for many plantspecies and many, and perhaps all, of the economically important plantspecies including corn plants.

Viruses such as the Cauliflower mosaic virus (CaMV) may also be used asa vector for introducing a transgene into plant cells (U.S. Pat. No.4,407,956). The CaMV viral DNA genome is inserted into a parentbacterial plasmid creating a recombinant DNA molecule which can bepropagated in bacteria. After cloning, the recombinant plasmid again maybe cloned and further modified by introduction of the desiredpolynucleotide sequence. The modified viral portion of the recombinantplasmid is then excised from the parent bacterial plasmid, and used toinoculate the plant cells or plants.

Other techniques, such as electroporation have also been used to maketransgenic plants. But fundamentally for the invention disclosed here,the particular technique of plant transformation does not matter. Oncethe plant has been genetically engineered, and a transgenic plant hasbeen created, the method of transformation of the original plant becomesirrelevant. A transgene inserted into the genome of one plant is thenfully inheritable by progeny plants of the original geneticallyengineered plant by normal rules of classical plant breeding. Forexample, in vegetatively propagated crops, the mature transgenic plantsare propagated by the taking of cuttings or by tissue culture techniquesto produce multiple identical plants. Selection of desirable transformedplants is made and new varieties are obtained and propagatedvegetatively for commercial use. In seed-propagated crops, the maturetransgenic plants can be self crossed to produce a homozygous inbredplant. The inbred plant produces seed containing the newly introducedtransgene. These seeds can be grown to produce plants that would producethe selected phenotype.

It should be understood that techniques of plant genetic engineeringhave been developed to the point where it is now practical to place anygenetic construct into almost any useful plant species including cornplants. The process does, however, still involve some random processes,most notably that insertions of foreign DNA into the genome of plantsstill occurs at random sites in the plant genome. As a result, in anygroup of plants emerging from a plant transformation process, theresults achieved for the different gene insertion events will vary,sometimes dramatically, depending on where the transgene is inserted.However, this variation does not mean stable results cannot be achieved,since the results tend to be consistent generation-to-generation foreach specific genetic insertion. One can also take advantage of thisvariation to generate lines with cornstarch characteristics changed todifferent degrees. As shown in the example below, when barley sbe IIawas used to generate transgenic corn plants, cornstarch from sometransgenic plants had lower gelatinization temperature andretrogradation rate while cornstarch from other transgenic plants hadhigher retrogradation rate. For those transgenic plants with cornstarchof lower gelatinization temperature and retrogradation rate, the degreeto which the gelatinization temperature and retrogradation rate werelowered were different.

The transgenic corn plants created by the methods described above, theseeds of which contain starch with changed characteristics, are withinthe scope of the present invention. Progeny and parts obtained from theplant such as seeds, are included in the invention, provided that theprogeny and the parts comprise cells that contain the transgene.

SBEs of different species provide starches obtained from these specieswith different characteristics. The present invention offers theopportunity to combine the advantages and avoid disadvantages ofstarches from two different species. The novel starches produced may addvalue to the corn crop for the existing uses, or create new products andnew markets for starch such as biodegradable plastics. When starch froma particular non-corn plant species has a desirable characteristicattributable to its SBE that is absent from cornstarches, apolynucleotide encoding a protein that has the SBE activity can be usedto generate transgenic corn plants from which one that produces starchwith the desired characteristic can be selected. The polynucleotide canbe the SBE gene itself. One of ordinary skill in the art will appreciatethat certain variations on the gene such as point mutations, insertionsand deletions will not abolish the specific SBE activity of the gene.Accordingly, these gene variants can also be used to generate atransgenic corn plants in the present invention. Given the potentialsilencing effect of an overexpression of the polynucleotide on allbranching enzyme activities, the same transgenic plants above can alsobe used to select for plants that produce starches with a highergelatinization temperature and retrogradation rate and for plants thatproduce starches with a high amylose content.

In one embodiment, a barley sbe IIa or a variant thereof that retainsthe specific SBE IIa enzymatic activity is used to generate transgeniccorn plants for producing cornstarch with lower starch gelatinizationtemperature and retrogradation rate or cornstarch with higher amylosecontent and retrogradation rate. Lower starch gelatinization temperatureand small enthalpy changes reduce energy consumption for starch cooking.Lower retrogradation rate of starch results in a better paste stability,and longer shelf life. It may also help retain water biding capacity andthickening power and results in superior paste properties as a sizingagent used in paper, textile, and other industries. High amylose starchhas many applications in the industry for its unique functionalproperties, and starches with high retrogradation rate may be used toproduce resistant starch for low caloric diet.

In other embodiments, other genes or a variant thereof are used forchanging cornstarch characteristics. Examples of these genes include butare not limited to barley sbeIIb (Genebank Accession Number AF064561),wheat sbe1A (Genebank Accession Number AF286318), wheat sbe1D (GenebankAccession Number AF286317), wheat sbeIIa-1 (Genebank Accession NumberY11282), wheat sbeIIa-2 (Genebank Accession Number U66376), wheat sbe2(Genebank Accession Number AF286319), wheat sbe as disclosed inWO0132886 (Genbank Accession Number AX134202), rice sbeI (GenebankAccession Number D11082), rice sbe3 (Genebank Accession Number D16201),pea sbeI (Genebank Accession Number X80009), pea sbeII (GenebankAccession Number X80010), a potato sbe, Arabidopsis sbe2-1 (GenebankAccession Number U11817), and Arabidopsis sbe2-2 (Genebank AccessionNumber U22428). Branching enzymes found in bacteria including Bacillusbacteria can also be used (e.g., Genebank Accession Numbers Z14057,Z25795, and AF008220). Sun et al. 1995 compares some of the genes listedabove and is herein incorporated by reference in its entirety.

The invention will be more fully understood upon consideration of thefollowing non-limiting example.

EXAMPLE

Materials and Methods

Construction of pMO5 vector: A plasmid 27.3-D was a kind gift from Dr.Brian Larkins' lab (University of Arizona), which contains the promoterof the maize gamma zein gene (Reina et al., 1990, which incorporatedherein by reference in its entirety) in a pUC19 backbone. A SacI-EcoRIfragment containing the Nos terminator of Agrobacterium tumefaciens wassub-cloned from pBI101.3 (Clontech, Palo alto, Calif.) into 27.3-D toform pMO1. Subsequently, the first intron of maize AdhI gene wasintroduced into pMO1 to derive pMO2. The full-length cDNA of maize Sulgene (James et al., 1995) was inserted into the NcoI-NotI sites of pMO2to create pMO5 (FIG. 1).

Construction of Barley SBE IIa Expression Vectors: For expression ofbarley SBE in corn, the barley sbeIIa cDNA (GenBank Accession NumberAF064560, provided as SEQ ID NO:1 herein) (Sun et al., 1998) wasamplified by PCR with Taq DNA polymerase using the upper primer(5′-ACGCGTAGATCTGGCGCCATGGCGGAAGTAAA-3′) (SEQ ID NO:3) and the lowerprimer (5′-CCCGGGTCTAGATTTTTTTTTTTTTTTTTT-3′) (SEQ ID NO:4). The PCRproduct was cloned into pTAg plasmid (R & D Systems, Minneapolis,Minn.). Primers BM1 (5′-ATCTGGATCCATGGCGGAAGTAAACA-3′) (SEQ ID NO:5) andBM2 (5′-GAGTATCCATCCGTATCTT-3′) (SEQ ID NO:6) were used to amplify a527-bp PCR fragment from the sbeIIa clone with Expand™ High Fidelity PCRsystem (Roche Molecular Biochemicals, Indianapolis, Ind.), and thefragment was digested by BamHI and was cloned into the BamHI site of aplasmid pMO5. The resulting plasmid was then cut with BamHI and SacI toaccept a 1.8 kb BamHI/SacI fragment of the sbeIIa clone. This plasmidwas further digested with SacI to accept a SacI/SacI fragment amplifiedfrom sbeIIa clone using primers BM4(5′-ATGCCCTTACAGAGCACCACCACCACCACCACTAAGAACCAGCAGCT-3′) (SEQ ID NO:7)and BM5 (5′-ATGTGAGAGCTCGGATGGTTCAGTGCAG-3′) (SEQ ID NO:8), resultingvector pMO11 (FIG. 2).

To add a 6×-histidine tag to the C-terminus of the barley sbeIIa clone,primer BM4 and BM5 were used to amplify a 295 bp PCR product from thebarley sbeIIa clone. The PCR fragment was further used as “megaprimer”to amplify a 701 bp PCR product from the sbeIIa clone with primer BM3(5′-TATGATAAATGCCGCCGTAGA-3′) (SEQ ID NO:9). The resulting PCR productwas digested with EcoRV and SacI restriction enzymes, and was clonedinto pMO11 cut with the same enzymes. The plasmid was then digested withSacI to accept a SacI/SacI fragment from the PCR product amplified byBM4 and BM5. The plasmid generated has a 6×-histidine tag just beforethe stop codon of barley sbeIIa cDNA and was named pMO12 (FIG. 2).Plasmids pMO11 and pMO12 were both sequenced to ensure that no mutationwas introduced and the restriction fragments ligated are in correctorientation.

Maize Transformation and Analyses: The maize transformation wasconducted using particle bombardment method at the Plant TransformationFacility of Iowa State University according to standard procedures(Fromm, 1994). Immature embryos of Hi II hybrid and Oh43 inbred wereused for the transformation. Gold particles coated with 1 μg plasmid DNAwere used to bombard embryogenic callus tissue with an instrument fromBiolistics (Ithaca, N.Y.). A Streptomyces bar gene on a separate plasmidwas co-bombarded as a selectable marker. The bar gene is expressedconstitutively from the maize ubiquitin promoter, with transcriptiontermination occurring from the A. tumefaciens 3′ nos region.Successfully transformed calli were identified by growth on mediacontaining the selective reagent Bialaphos at 3 mg/L. Approximately 20clones (independent transgenic events) for each construct wereregenerated into Plants. Primary regenerated plants (R0) were outcrossedto both Oh43 inbred and Oh43 with homozygous amylose extender (ae)mutant alleles.

DNA was extracted from callus tissue and leaves of regenerated plants byusing the method of Dellaporta (1994) (incorporated by reference in itsentirety) with the following modifications. The extraction buffercontained 100 mM Tris, pH 8.0, 50 mM EDTA, pH 8.0, 500 mM NaCl, 10 mMbeta-mercaptoethanol, and 1% SDS. Callus tissue was ground in theextraction buffer in the presence of about 10 mg carborundum (FisherScientific, Pittsburgh, Pa.) instead of liquid nitrogen. DNA wasdissolved in 10 mM Tris-HCl, pH 8.0. An RNase treatment was added to theDNA isolation protocol as follows. The DNA isolated was treated withRNase A (Sigma, St. Louis, Mo.) at 100 μg/ml for 15 min at 37° C. Then 1μl of 5 M NaCl per 20 μl DNA and three volumes of 100% ethanol wereadded. After 10 min on ice, samples were spun at 13,000 g for 10 min.The pellet was washed with 70% ethanol, air or vacuum dried, andresuspended in 10 mM Tris-HCl (pH 8.0).

PCR analysis was used to screen the callus clones and confirm thepresence of intact copies of transgenes in the regenerated plants. Twosets of primers (FIG. 2) were used to amplify the transgene productswith Expand™ Long Template PCR System (Roche Molecular Biochemicals,Indianapolis, Ind.) that is composed of a mixture of Taq and Pwo DNApolymerases. Primer, puc is a 23-mer (5′-GTGTGGAATTGTGAGCGGATAAC-3′)(SEQ ID NO:10), zein1 is a 24-mer (5′-TGAGCCACGCAGAAGTACAGAATG-3′) (SEQID NO:11), and nos is a 22-mer (5′-ATCATCGCAAGACCGGCAACAG-3′) (SEQ IDNO:12). Manufacturer's buffer 1 and 500 ng of template DNA were includedin the reaction mixtures. The PCR reactions were run in a Mastercycler®personal thermocycler (Eppendorf Scientific, New York, N.Y.) with aninitial denaturation for 2 min at 94° C.; followed by 30 cycles of 94°C. for 20 s, 65° C. for 30 s, and 68° C. for 2 min 20 s (zein1 and nosprimer pair) or 3 min (puc and nos primer pair). From the 11th cycle,each cycle had 20 s more elongation time at 68° C. than the previouscycle. The reaction was concluded with a prolonged elongation time of 7min at 68° C. A 3.4 kb or 4.4 kb PCR fragment of barley SBEIIa transgenewas robustly amplified. The fragments cover the length of the entiretransgene (FIG. 2).

Assay of Branching Enzyme Activity: Kernels were isolated fromdeveloping ears 20 days after pollination and were immediately frozen inliquid nitrogen before being stored in freezer. Individual seeds wereground in a tube containing 2.5 ml/g extraction buffer (50 mM Tris-HCl,pH 7.0; 10% glycerol; 10 mM EDTA; 5 mM DTT, and 1% proteinase inhibitorcocktail (#P-9599) from Sigma) using a hand drill connected with amicrofuge pestle (Kontes Sci., Vineland, N.J.; item number 749520) andvortexed. The homogenate was spun at 10,000 g for 10 min at 4° C. andthe supernatant was saved. Protein concentration was determinedaccording to the method of Bradford (1976) using a Bio-Rad Protein AssayKit with BSA as the standard.

Branching enzyme activity was measured by thephosphorylation-stimulation assay (Sun et al., 1997, incorporated byreference in its entirety) using 20 μg seed protein. The amount ofbranching enzyme was in the linear range with the rate of phosphaterelease. A control with 20 μg of boiled yeast protein was alsoincubated. The small amount of Pi produced in the control was subtractedfrom those produced in samples with active branching enzyme present.

Isolation and Analyses of Starch: Mature seeds were weighed and steepedin 0.45% sodium metabisulfite for 48 hrs at room temperature. Singleseed was homogenized in 10 ml water with a Ultra-Turrax T25 homogenizer(Janke & Kunkel, Staufen, Germany) for 1 min, after seed coat and embryowere removed. The homogenate was centrifuged at 50,000 g for 30 min toseparate the soluble material in the supernatant and starch in thepellet. The starch-containing pellet was resuspended in about 200 mlwater, passed through a 53 μm nylon mesh filter (Spectrum, Laguna Hills,Calif.) into a beaker. The starch was sedimented at 4° C. for 2 hrs andthe supernatant was decanted. The sediments were then stirred up with200 ml fresh water. The sedimentation and decanting were performed fourtimes. After last decanting, the starch was dried at room temperatureand weighed.

Gelatinization and retrogradation properties of starches were analyzedby using a differential scanning calorimeter (DSC-1, Perkin-Elmer,Norwalk, Conn.) equipped with an intracooling II system as described byJane et al. (1999), which is incorporated by reference in its entirety.The data were averages of three replicates of each starch sample.Analysis of variance was used for data analysis (Steel and Torrie,1960). Mean differences were compared by using least significantdifferences (LSD) at both the 5% and 1% levels of probability.

Results

Callus clones were screened for the presence of transgenes by PCR usingprimers that cover the entire length of the transgene (FIG. 2). Mostclones had the intact copy of the transgene, but some clones had notransgene or only rearranged/truncated copies. Only those clones withintact transgene copies were selected for regeneration into plants. Wehave obtained a total of 31 clones (independent transgenic events). Thepresence of intact transgenes in regenerted plants was confirmed by PCRanalyses of leaf DNA.

Most regenerated plants look normal. These R₀ plants, mostly as females,were outcrossed to both OH43 inbred and aeae mutant in OH43 inbredbackground to get R₁ seeds. Of the 31 clones we obtained, 11 showedvarious degree of translucent and shrunken kernel phenotype in abouthalf of the seeds on an ear, especially when crossed to an aeae maleplant.

Starches were isolated from single R₁ seeds and their thermal propertieswere measured by differential scanning calorimetry (DSC). Results fromsome selected clones are presented in Table 1. Starches of cloneP90-4×OH43 inbred displayed lower onset gelatinization temperatures thanstarches from the untransformed control. Their thermal transition peakof starch gelatinization initiated at a substantially lower temperatureof about 55° C., compared with above 60° C. for the untransformedcontrol. Starches isolated from the slightly shrunken kernels of cloneP90-4×OH43 displayed high completion temperatures and large ranges ofgelatinization temperature. The wide peaks of the thermal profiles forthese starches resemble that of aeae mutant or high amylose starches(Wang et al., 1992; Kasemsuwan et al., 1995; Jane et al., 1999). Thesekernel phenotypes and DSC profiles were also observed in crosses ofP90-4 to aeae mutant and were not found in untransformed controlcrosses. The results indicated that these features were not likelycaused by the ae mutant allele but were resulted from the transgenes.Starches from some seeds of clones P90-4, P90-8 and P90-15 hadsubstantially smaller enthalpy change than the untransformed controls,and the value for a seed from P90-2×OH43 cross was only 30% of thecontrols.

All the clones we analyzed had some seeds with lower starchretrogradation rate than the untransformed controls (Table 1). On theother hand, starches from some seeds showed much higher retrogradationrate than the controls, sometimes reaching 100%. Their retogradationprofiles are similar to that of aeae mutant or other high amylose maizestarches as reported by others (Kasemsuwan et al., 1995; Jane et al.,1999).

Branching enzyme activities showed a wide range of variations, rangingfrom almost no activity to double of the untransformed controls (Table1). Without intending to be limited by theory, we hypothesize that thesevariability may explain some of the variations in thermal propertiesdisplayed by some seeds. Expression of the barley sbe IIa gene may lowerthe gelatinization temperature and retrogradation rate, making thecornstarch function more like barley starch. When the expression of thebarley BE gene exceeds certain threshold level, however, it may causesilencing of all BE genes. Gene silencing effect may reduce the BEactivities in the kernel and lead to the production of high-amylosestarch like aeae mutant. It is well established that overexpression oftransgenes may lead to gene silencing. Linked and unlinked copies oftransgenes and related endogenous genes in plants can be epigeneticallysilenced by homology-based mechanisms that operate at either thetranscriptional or post-transcriptional level (Matzke et al., 1996;Matzke and Matzke, 1998). All SBEII gene family members from wheat,barley, maize, rice, pea, and Arabidopsis share a high degree of aminoacid identity (90%–95%) in the major portion of their coding sequences(Sun et al., 1998). Silencing of the granule-bound starch synthase I(GBSSI) in potato has been achieved by introduction of either antisenseor sense copies of GBSSI cDNA or genomic DNA (Wolters and Visser, 2000).Compared with crosses with OH43 inbred, kernels from crosses with aeaemutant have reduced Ae (endogenous SBEIIb) allele in their endosperm andthus reduced competition with the barley SBEIIa transgene. That mayallow the barley BE gene to have more pronounced effect. Indeed, mosttranslucent and sunken kernel phenotype was observed in crosses withaeae mutant.

A wide range of genetic variation in starch BE activities and thermalproperties has been observed in the corn kernels transformed with barleySBEIIa gene. Lower starch gelatinization temperature and small enthalpychanges reduce energy consumption for starch cooking. Lowerretrogradation rate of starch results in a better paste stability, andlonger shelf life. It may also help retain water biding capacity andthickening power and results in superior paste properties as a sizingagent used in paper, textile, and other industries. High amylose starchhas many applications in the industry for its unique functionalproperties, and starches with high retrogradation rate may be used toproduce resistant starch for low caloric diet.

The present invention is not intended to be limited to the foregoingexample, but encompasses all such modifications and variations as comewithin the scope of the appended claims.

TABLE 1 Thermal properties of starches measured by differential scanningcalorimetry and branching enzyme (BE) activities of R₁ kernels fromindependent transgenic clones Kernel Starch Gelatinzation^(c) StarchRetrogradation^(d) BE Specific Kernel weight T_(o) T_(p) T_(c) Range ΔHT_(o) T_(p) T_(c) ΔH R activity^(e) Genotype^(a) phenotype^(b) mg ° C. °C. ° C. ° C. J/g ° C. ° C. ° C. J/g % % of control Hill untran. control× OH43 N 212.3 68.0 71.7 75.7 7.7 12.0 37.9 49.4 61.9 6.8 56.4 95.9 N236.4 67.8 70.8 74.3 6.5 12.7 39.3 49.8 62.1 7.0 55.4 95.9 N 150.1 68.071.9 76.0 8.0 12.4 38.7 49.5 62.3 7.3 59.2 84.2 N 135.0 67.6 71.4 75.47.8 12.4 40.1 50.3 62.4 7.8 62.8 124.0 P90-2 × OH43 N 328.4 66.9 71.775.1 8.2 11.9 42.0 52.7 62.4 5.6 47.5 9.4 N 232.5 70.1 73.1 76.2 6.112.6 42.9 54.1 63.2 6.2 49.3 44.4 N 271.0 69.0 74.2 82.2 13.2 3.8 47.557.0 63.6 1.4 36.4 4.7 N 318.4 68.1 71.9 75.3 7.2 11.3 42.5 52.7 61.55.6 49.3 21.1 P90-4 × OH43 N 337.6 64.0 (55.7) 70.4 75.0 11.0 11.9 38.352.2 60.9 6.2 52.0 233.9 N 219.6 66.3 (55.3) 70.5 74.7 8.4 12.5 41.051.7 61.9 5.5 44.4 212.9 SS 230.1 66.5 (55.1) 72.4 88.9 22.4 14.3 41.756.4 111.0 47.8 100.0 84.2 SS 283.6 66.1 (55.1) 72.7 102.8 36.7 11.939.9 54.9 104.7 26.9 100.0 44.4 P90-8 × OH43 N 169.8 67.4 71.5 77.2 9.812.9 42.1 52.7 61.8 6.8 52.3 117.0 N 167.3 68.4 72.0 76.5 8.1 13.6 42.153.1 61.6 7.3 54.0 121.6 SS 149.1 67.4 72.5 79.7 12.3 8.0 44.1 53.7 63.53.1 37.7 84.2 SS 141.4 67.0 72.7 81.0 14.0 7.5 44.6 56.5 63.6 2.6 34.6161.4 Hill untran. Control × N 228.1 67.5 72.7 77.3 9.8 13.6 40.8 52.262.7 7.5 55.4 108.0 OH43aeae N 253.6 68.0 72.4 76.9 8.9 12.8 41.7 52.362.4 7.7 60.0 98.4 N 234.2 68.2 72.5 76.9 8.7 12.0 38.1 49.8 61.9 7.563.1 109.1 N 251.0 68.0 72.7 77.2 9.2 12.6 38.8 49.8 63.0 8.0 64.0 84.5P90-4 × OH43aeae N 209.3 66.9 71.5 76.3 9.4 11.2 39.3 50.5 62.6 8.2 73.4102.7 N 285.2 66.0 72.7 77.1 11.1 12.5 40.4 52.1 62.3 7.7 61.5 105.9 SS176.5 68.1 (55.6) 87.3 103.0 34.9 10.3 44.3 91.0 103.7 13.3 100.0 103.7SS 213.9 68.8 88.2 102.5 33.7 9.0 78.8 93.1 101.2 4.5 61.4 110.2 P90-8 ×OH43aeae N 129.0 68.7 72.3 76.3 7.6 12.8 40.6 52.3 62.4 7.9 61.8 51.3 N108.7 70.1 73.3 77.2 7.1 13.3 41.7 52.8 63.2 9.2 64.0 89.8 S 90.8 67.2(53.2) 73.7 82.7 15.5 11.5 44.0 53.7 62.2 1.6 13.9 44.9 50.3 P91-15 ×OH43aeae N 142.3 68.7 73.5 78.4 9.7 8.5 43.8 54.5 63.7 5.2 60.5 90.9 N139.7 68.8 73.4 78.1 9.3 9.6 39.8 53.8 63.5 3.5 37.1 46.0 N 153.5 68.274.1 81.0 12.8 8.8 42.5 57.4 68.0 3.2 35.8 93.0 N 173.0 68.7 73.3 78.09.3 9.2 42.5 54.4 64.9 4.2 45.8 26.7 LSD_(0.05) .9 .5 1.2 1.4 1.4 2.31.2 1.3 1.4 7.4 11.8 LSD_(0.01) 1.2 .7 1.6 1.9 1.9 3.0 1.6 1.7 1.9 9.815.0 ^(a)Crosses are shown as female × male. Hill untran. control,tissue-culture derived untransformed control in Hill background; OH43,OH43 inbred; OH43aeae, aeae mutant in OH43 background. ^(b)N normal; SS,slightly shrunken; S, shrunken. ^(c)T_(o), onset temperature; T_(p),peak temperature; T_(c), completion temperature; ΔH, enthalpy change.Range is T_(c)–T_(o). Thermal transition initiation temperatures are inparenthesis. ^(d)% R, percentage of retrogradation. Values of starchgelatinization and retrogradation are averages of three replicates.^(e)Specific activities of BE for crosses to OH43 are presented aspercentage of the mean value of (Hill untran. control × OH43); and thespecific activities for crosses of OH43aeae are shown as percentage ofthe mean value of (Hill untran. control × OH43aeae). Values are averagesof two replicates.

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1. A transgenic corn plant, corn plant cell, or corn plant tissuecomprising: a polynucleotide transgene that encodes barley starchbranching enzyme (SBE) IIa comprising SEQ ID NO:2 wherein the expressionof barley SBE IIa in the transgenic corn plant changes a cornstarchcharacteristic as compared to a plant of the same genetic backgroundwithout the polynucleotide transgene.
 2. The transgenic corn plant, cornplant cell, or corn plant tissue of claim 1 wherein the polynucleotidehas the nucleotide sequence of nucleotide 7 to nucleotide 2208 of SEQ IDNO:1.
 3. The transgenic corn plant of claim 1 wherein seeds grown on thetransgenic plant contain cornstarch having at least one of thecharacteristics of lower gelatinization temperature, lowerretrogradation rate, higher gelatinization temperature, higherretrogradation rate, or higher amylose content in comparison tocornstarch from seeds of a non-transgenic corn plant of the same geneticbackground and being grown under the same conditions.
 4. The transgeniccorn plant of claim 3, wherein seeds grown on the transgenic plantcontain cornstarch having at least one of the characteristics of lowergelatinization temperature or lower retrogradation rate.
 5. Thetransgenic corn plant of claim 3, wherein seeds grown on the transgenicplant contain cornstarch having at least one of the characteristics ofhigher gelatinization temperature or higher retrogradation rate.
 6. Aseed derived from the transgenic corn plant of claim 1 wherein the seedcomprises the polynucleotide transgene.
 7. The seed of claim 6 whereinthe seed contains cornstarch having at least one of the characteristicsof lower gelatinization temperature, lower retrogradation rate, highergelatinization temperature, higher retrogradation rate, or higheramylose content in comparison to cornstarch from a seed of anon-transgenic corn plant.
 8. The seed of claim 7 wherein the seedcontains cornstarch having at least one of the characteristics of lowergelatinization temperature or lower retrogradation rate.
 9. The seed ofclaim 7 wherein the seed contains cornstarch having at least one of thecharacteristics of higher gelatinization temperature or higherretrogradation rate.
 10. A method for making a transgenic corn plant ofclaim 1 comprising the steps of: contacting a corn plant cell with anucleic acid comprising a polynucleotide encoding barley SBE IIacomprising SEQ ID NO:2; identifying a plant cell carrying thepolynucleotide; regenerating a transgenic plant from the plant cellcarrying the polynucleotide; and determining whether seeds from thetransgenic plant contain cornstarch having at least one characteristicchanged in comparison to cornstarch from seeds of a non-transgenic cornplant of the same genetic background and being grown under the sameconditions.
 11. A method for making a transgenic corn plant of claim 1comprising the steps of: randomly inserting a nucleic acid comprising apolynucleotide encoding barley SBE IIa comprising SEQ ID NO:2 into thegenome of a corn plant; and selecting plants having seeds that containcornstarch having at least one characteristic changed in comparison tocornstarch from seeds of a non-transgenic corn plant of the same geneticbackground and being grown under the same conditions.
 12. The method ofclaim 10 or claim 11, wherein the polynucleotide has a nucleotidesequence of nucleotide 7 to nucleotide 2208 of SEQ ID NO:1.
 13. Themethod of claim 10 or claim 11, wherein the changed characteristic ofcornstarch is gelatinization temperature, retrogradation rate, oramylose content.
 14. The method of claim 10 or claim 11, wherein thecornstarch with a changed characteristic has a lower gelatinizationtemperature, lower retrogradation rate or both.
 15. The method of claim10 or claim 11, wherein the cornstarch with a changed characteristic hasa higher gelatinization temperature, higher retrogradation rate or both.